SiRNA
Overview Small Interfering RNA (siRNA) is sometimes referred to as Short Interfering RNA or Silencing RNA. Small interfering RNA has a variety of roles, but is most often used in the RNA interference pathway. siRNAs are a double stranded RNA molecule that are approximately 20-25 basepairss long, and are most often used in the RNA interference pathway. The siRNA terminates the expression of mRNA molecules of specific genes, by binding to a complementary mRNA sequence. The ability of siRNA to bind complementarily to mRNA allows for extreme specificity silencing the expression of particular genes. The siRNA binding to the mRNA after transcription, results in the breakdown and degredation of the mRNA before it is able to be translated. This role of siRNA was first reported in Science in 1999. The structure of siRNAs is fairly uniform; they are double stranded RNA molecules that are 20 to 25 base pairs long and have a phosphorylated 5' end and a hydroxylated 3' end, as well as 2 overhanging nucleotides. Dicer enzymes are responsible for the catalysis of siRNAs using long dsRNAs and small hairpin RNAs. siRNAs can also be introduced to a mammalian cell by the process of transfection, which is outlined in the image below. History The post-transcriptional gene silencing of siRNA in plants was discovered by David Baulcombe and colleagues through their work at Sainsbury laboratory in Norwich, England. Baulcombe's findings were reported in Science in 1999. Soon after, Thomas Tuschl and colleagues reported that synthetic siRNA could induce RNA interference in mammalian cells in Nature. Baulcombe's laboratory studied the mechanism of how post transcriptional gene silencing occurs in plants that have been transformed with foriegn or endogenous DNA. PTGS also results in the reduced accumulation of RNA molecules with sequence similarity to the silenced DNA. It was also noted that double stranded RNA induces the same results in nematodes, insects, and protozoa. Due to the high sequence specificity of PTGS it was thought that large complementary sequences of RNA must bind to the mRNA and target it for degredation. However, if this was the case large molecules of RNA that is complementary to the mRNA should be able to be detected with common polyacrylamide gel electrophoresis. Large complementary RNA molecules were not found in organisms that exhibited PTGS. A method of low molecular weight RNA analysis was used to detect small RNA moleculed in plants that exhibited four classes of PTGS. One class of PTGS studied was trangene-induced PTGS. This was studied in five lines of tomato plant. Two of the five lines of tomato plant exhibited PTGS, and in these two lines small Interfering RNA of about 25 nucleotides in length were discovered, by polyacrylamide gel electrophoresis. Uses siRNA can be used to a variety of different ends. Post transcriptional gene silencing is useful in many areas of research. Testing Hypotheses for Gene Function. In determining gene function siRNA is used to specifically silence the gene of interest post transcriptionally. This is useful in determining the gene's function because if the gene is silenced the role that gene should be altered in a predictable way. Pathway Analysis siRNA is used in pathway analysis by silencing a gene in a pathway and then observe the implications of that silencing. An example of this would be silencing of a transcription factor. Once the transcription factor has been silenced the genes that the transcription factors affect can no longer be stimulated. Using this information the function of many genes in a pathway can be determined. Gene Therapy The use of siRNA in gene therapy is a growing area of research. However, it is not used practically as of yet but publications have shown promising results. One such publication studied the use of siRNAs in inhibiting the replication of HIV. References Hamilton, Andrew J., and David C. Baulcombe. "Go to Science." Science Magazine: Sign In. Science, n.d. Web. 29 Sept. 2014. . "Small Interfering RNA." Wikipedia. Wikimedia Foundation, 17 Sept. 2014. Web. 29 Sept. 2014. . "RNAi as a Tool for Mammalian Gene Analysis: Applications of SiRNAs."Applications of SiRNAs. Life Technologies, 2014. Web. 01 Oct. 2014. .